Unveiling a novel exopolysaccharide produced by Pseudomonas alcaligenes Med1 isolated from a Chilean hot spring as biotechnological additive

Abstract

Exopolysaccharides (EPSs), a constitutive part of bacterial biofilm, act as a protecting sheath to the extremophilic bacteria and are of high industrial value. In this study, we elucidate a new EPS produced by thermotolerant (growth from 34-44 °C) strain Pseudomonas alcaligenes Med1 from Medano hot spring (39.1 °C surface temperature, pH 7.1) located in the Central Andean Mountains of Chile. Bacterial growth was screened for temperature tolerance (10-60 °C) to confirm the thermotolerance behaviour. Physicochemical properties of the EPS were characterized by different techniques: Scanning Electron Microscopy- Energy Dispersive X-ray Spectroscopy (SEM-EDS), Atomic Force Microscopy (AFM), High-Performance Liquid Chromatography (HPLC), Gel permeation chromatography (GPC), Fourier Transform Infrared Spectroscopy (FTIR), Nuclear Magnetic Resonance (NMR), and Thermogravimetric analysis (TGA). Whole genome of P. alcaligenes Med1 has also been studied in detail to correlate the structural and functional characteristics with genomic insight. The EPS demonstrated amorphous surface roughness composed of evenly distributed macromolecular lumps composed of mainly carbon and oxygen. The monosaccharide analysis has shown the presence of glucose, galactose, and mannose sugars at different ratios. TGA revealed the high thermal stability (315.3 °C) of the polysaccharide. The GPC has shown that Med1 is a low molecular weight polysaccharide (34.8 kDa) with low PI. The 2D-NMR linkage analysis suggests a diverse array of glycosidic bonds within the exopolysaccharide structure. The functional properties of the EPS were evaluated for food industry applications, specifically for antioxidant (DPPH, FRAP an H₂O₂). Extracted Med1 EPS revealed significant emulsification activity against different food grade vegetative oils (Coconut oil, Corn oil, Canola oil, Avocado oil, Sunflower oil, Olive oil, and Sesame oil). The highest 33.9% flocculation activity was observed with 60 mg L^-1 EPS concentration. It showed water-holding (WHC) of 107.6% and oil-holding (OHC) capacity of 110.8%. The functional EPS produced by Pseudomonas alcaligenes Med1 from Central Andean Chilean hot spring of central Chile can be a useful additive for the food-processing industry.

Keywords: Pseudomonas; Additive; Biotechnology; Exopolysaccharide; Food industry.

Fig. 1

Map showing the location of Medano hot spring located in the Andean Mountains in the Maule region, Chile, and photographs of the study site. On the right, the maximum likelihood phylogeny of isolate Med1 (marked with red arrow) shows similarity with Pseudomonas alcaligenes. On top right corner SEM micrograph demonstrating the morphology of the isolated Med1.

Fig. 2

(A) Phylogenetic tree showing maximum similarity within the species (B) Circular representation of annotated whole genome of Pseudomonas alcaligenes Med1 (C) Pairwise ANI% value-based heatmap showing maximum similarity within the species.

Fig. 3

Circular genomic map of Med1 obtained from PHASTEST.

Fig. 4

Demonstrating Genomic Islands predicted from the genome of Pseudomonas alcaligenes Med1.

Fig. 5

Biosynthetic Gene Cluster Analysis of Pseudomonas alcaligenes Med1.

Fig. 6

Relative distribution of pan, core and accessory genome through Spine and Agent tool plotting each category or subcategory.

Fig. 7

Response surface plot (2-D) interaction effect expressed, respectively, in X1 and X2 axes. Here, inputs are 30 experimental runs carried out under conditions established by CCD matrix. EPS production as function with (A) nitrogen source (g L⁻¹) and carbon source (g L⁻¹) (B) carbon source (g L⁻¹) and pH (C) bacterial concentration and carbon source (mL L⁻¹) (D) nitrogen source (g L⁻¹) and pH (E) nitrogen source (g L⁻¹) and bacterial concentration (mL L⁻¹) and (F) pH and bacterial concentration (mL L⁻¹).

Fig. 8

(A) Normal plot of residuals; (B) Predicted versus actual plot for EPS production and (C) Ramp chart for optimization conditions of EPS production by Med1, developed by Design Expert software.

Fig. 9

Surface morphology of the EPS produced by Pseudomonas alcaligenes Med1, where (A) ×3000 (B) ×5000 respectively; and (C) energy dispersive X-ray spectroscopy (EDS) (D) 2D; and (E) 3D AFM images, and (F) size distribution patterns.

Fig. 10

(A) HPLC analysis of the constituting monosaccharides of the Med1 EPS. (B) Thermogravimetric curves of Med1 exopolysaccharide (C) FTIR-ATR spectra of Med1 polysaccharide.

Fig. 11

(A)¹H NMR spectra, (B) HSQC ¹³C/¹H NMR spectrum, and (C) probable structure of a new galactoglucomannan as the main exopolysaccharide produced by thermotolerant Pseudomonas alcaligenes Med1. The structure was created following the symbol nomenclature for glycans guidelines and respecting the percentage quantity of each monosaccharide found after integrating the anomeric carbon regions.

Fig. 12

In vitro antioxidant capability of the EPS produced by Pseudomonas alcaligenes Med1; where (A) ABTS radical scavenging activity; (B) H₂O₂ radical scavenging activity; (C) Ferric reducing ability (D) Emulsification activity against different food grade vegetative oil of the EPS and (E) Bioflocculation activity Pseudomonas alcaligenes Med1.